Acknowledgment
This work was supported in part by NIH/NCI grants R01CA160331 to R.Z., R01CA131582 to D.W.S., and an institutional grant to The Wistar Institute (NCI Cancer Core Grant CA010815).
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_13, © Springer Science+Business Media New York 2017
Chapter 13
Detection of Reactive Oxygen Species in Cells Undergoing Oncogene-Induced Senescence
Rabii Ameziane-El-Hassani and Corinne Dupuy
Abstract
Reactive oxygen species (ROS) derive from molecular oxygen and present higher reactivity. ROS designa- tion comprehends free radicals such as superoxide radical (O 2 ° − ), hydroxyl radical (OH° − ); but also non- radical molecule like hydrogen peroxide (H 2 O 2 ). ROS play a critical role in several physiological functions like proliferation and signalling pathways. Thanks to cellular (oxidant/antioxidant) systems, ROS level is tightly regulated to avoid excessive damage to biological macromolecules (proteins, lipids, and DNA). An imbalance of redox equilibrium can lead to persistent oxidative stress favoring senescence, infl ammation, and carcinogenesis.
Oncogene activation can induce severe or irreparable DNA damage and causes proliferative arrest named senescence. Senescence acts as a tumorigenesis barrier, and its bypass can promote transition between normal homeostasis and neoplastic transformation. The mechanisms through which oncogenes induce senescence remain unclear but involve increased cellular level of Reactive Oxygen Species. Among ROS, H 2 O 2 is of particular interest because the hydrogen peroxide is more stable, can diffuse actively or freely through the cellular membranes, and can generate locally the hydroxyl radicals by iron-mediated Fenton reaction. Interestingly, growing data support the role of H 2 O 2 in the propagation of the stressful effects of senescent cells to their neighbors through the bystander effect. In this protocol, we present our routinely used methodology to detect extracellular H 2 O 2 using the Amplex red/horseradish peroxidase assay. This highly sensitive method detects specifi cally H 2 O 2 , and offers the possibility to quantify it using the H 2 O 2 standard curve.
To illustrate this method of detection of extracellular H 2 O 2 in cells undergoing oncogene-induced senescence, we compare two human cell lines: BCPAP (from human papillary thyroid carcinomas carrying BRAF V600E mutation) and HTori-3.1 cell line (immortalized human thyroid epithelial cells) because expres- sion of BRAF V600E in human thyroid cells triggers senescence.
Key words Reactive oxygen species , Hydrogen peroxide , Amplex red , Senescence
1 Introduction
Because of its stability and membrane permeability, H 2 O 2 is usually chosen to quantitate the ROS production by cells. Chromogenic and fl uorogenic substrates are currently used to measure H 2 O 2 , but assays using fl uorogenic substrates are more sensitive than
assays that use chromogenic substrates [ 1 , 2 ]. Several substrates are used for the fl uorometric detection of H 2 O 2 based on the oxida- tion of each substrate by H 2 O 2 in the presence of the peroxidase, and the monitoring of the fl uorescence intensity. The 2′,7′-dichlo- rodihydrofl uorescein diacetate (H 2 DCF-DA), Scopoletin (7-hydroxy-6-methoxy-coumarin), Homovanillic acid (4-hydroxy- 3-methoxy-phenylacetic acid), and the Amplex Red ( N -Acetyl- 3,7-dihydroxyphenoxazine) are widely used to detect H 2 O 2 , but among them the Amplex Red remains the best sensor of H 2 O 2 due to its high sensitivity and specifi city [ 1 , 2 ]. In fact, (1) H 2 DCF-DA widely used to detect intracellular H 2 O 2 can be also oxidized by superoxide anion and nitric oxide, (2) Scopoletin, which is specifi c for extracellular H 2 O 2 is barely stable and its fl uorescence is highly dependent on pH and temperature, and (3) Homovanillic acid is specifi c for extracellular H 2 O 2 , more stable and more sensible than scopoletin but less sensible than Amplex Red [ 1 , 2 ].
The Amplex Red is a nonfl uorescent molecule that when oxi- dized by H 2 O 2 in the presence of HRP (horseradish peroxidase) generates resorufi n, a highly red-fl uorescent product [ 1 , 2 ]. This probe presents low background fl uorescence, and in the presence of peroxidase, the Amplex Red reacts with H 2 O 2 in a 1:1 stoichi- ometry to produce the resorufi n. Fluorescence can then be mea- sured with a fl uorescence microplate reader using excitation at 530 ± 12.5 nm and fl uorescence detection at 590 ± 17.5 nm.
This protocol can easily be used on any cell culture system to detect the extracellular H 2 O 2 under appropriate experimental con- ditions [ 3 – 6 ]. The Amplex Red assay also allows the measure of the H 2 O 2 in the cellular particulate fractions [ 5 , 6 ]. When H 2 O 2 is in excess, Amplex Red reagent is an ultrasensitive assay for peroxi- dase activity [ 6 ].
2 Materials